Composition method for determining the presence of leukocyte cells, esterase or protease in a test sample

ABSTRACT

A composition and method for determining the presence of leukocyte cells, esterase or protease in a test sample are disclosed. The composition contains a lactate ester having the structure ##STR1## wherein A is an alcohol blocking group, and wherein B-O- is a residue of compound B-OH and provides a detectable response when the lactate ester is hydrolyzed; and a buffer. One class of lactate ester has the structure ##STR2## wherein X is O, S or NR 2 , R is aryl or lower alkyl, R 1  is hydrogen or lower alkyl, and R 2  is hydrogen, lower alkyl or aryl.

FIELD OF THE INVENTION

The present invention relates to a novel composition, test device andmethod of assaying a test sample for the presence of leukocyte cells,esterase or protease. More particularly, the present invention relatesto using a lactate ester, like a hydroxy-protected5-phenyl-3-hydroxy-pyrrolyl-(L)-lactate ester, as a chromogenicsubstrate for a serine protease-based enzyme. The lactate esters undergoa detectable or measurable response upon contact with a test samplecontaining leukocyte cells, esterase, elastase or protease. Acomposition comprising a lactate ester and a diazonium salt couplingagent provides a more sensitive assay for leukocyte cells, esterase orprotease, therefore improving detection of leukocyte cellconcentrations. The increased sensitivity demonstrated by a combinationof the lactate esters of the present invention and a diazonium saltcoupling agent provides an improved method of assaying for leukocytecells, esterase or protease in a test sample, such as a biologicalfluid, like urine. The lactate esters are particularly useful indetecting leukocyte cell concentrations in a test sample by a simpledip-and-read method using a dry phase test strip which incorporates alactate ester of the present invention.

BACKGROUND OF THE INVENTION

The presence of an abnormally high level of leukocyte cells in the urineof an individual is indicative of a pathological condition, such as akidney or urogenital tract infection. The detection of leukocyteesterase in urine is an indirect test for bacteriuria (i.e., anabnormally high level of bacteria), and therefore infection.Accordingly, an accurate urine leukocyte cell assay, or leukocyteesterase assay, is valuable to a physician in the diagnosis andtreatment of kidney and urogenital tract infections.

Traditionally, technicians relied upon visual techniques to countleukocyte cells either in urine sediment or in uncentrifuged urine. Theconventional visual technique requires expensive equipment, such as acentrifuge and microscope, in addition to an inordinate amount oftechnician time. An additional disadvantage of the visual technique isthat only intact leukocyte cells can be counted. However, leukocytecells in the urinary system are subject to extensive cell lysis. Forexample, in a urine having an abnormally high pH, the half life of aleukocyte cell can be as low as 60 minutes. Since lysed leukocyte cellsescape detection in the visual technique, an erroneously low, or a falsenegative, result for leukocyte cells can result.

A visual examination for leukocyte cells in urine can be performed onuncentrifuged urine or on urine sediment. The latter method requirescentrifuging the urine sample, isolating the sediment, then visuallyinspecting the sediment, wherein the technician counts the number ofleukocyte cells appearing in the viewing field. This visual technique iscomplicated by the presence of other components in the urine sediment,such as epithelial cells and salt particles. The presence of variousurine sediment constituents, coupled with other factors, like anonhomogeneous sample or differing optical powers between microscopes,can lead to substantial assay errors.

Therefore, a quick, easy method of assaying for leukocyte cells, whicheliminates the need for time-consuming counting techniques and expensiveequipment, and which provides an accurate assay for intact and lysedleukocyte cells, would constitute a significant advance in the art. Thepresent invention provides such an advance. Moreover, because thepresent invention is based on the enzymatic activity of esterase orprotease in leukocyte cells, and not on the ability to visually observeand count intact leukocyte cells, the method is free of the assayinaccuracies described above.

Prior to the present invention, compositions and methods of determiningthe presence or concentration of leukocyte cells, esterase or proteasein a test sample utilized chromogenic esters which produced an alcoholproduct as a result of hydrolysis by an esterase or protease. The intactchromogenic ester has a color different from the alcohol hydrolysisproduct. The color change generated by hydrolysis of the chromogenicester therefore provides a method of detecting the presence orconcentration of esterase or protease, which in turn is correlated tothe presence or concentration of leukocyte cells. Many of these priorcompositions used accelerator compounds and diazonium salt couplingagents in conjunction with the chromogenic ester to improve assayresponse.

A present day urine assay for leukocyte esterase, and thereforeindirectly for leukocyte cells, is a dry phase test strip termedLEUKOSTIX®, available from Miles, Inc., Elkhart, Ind. This test stripdetects esterase activity released from granules of leukocyte cells intothe urine. The released esterase is termed "human leukocyte elastase"(HLE), and is a serine protease-based enzyme.

In the assay for leukocyte esterase or HLE using a LEUKOSTIX® strip, theenzyme hydrolyzes a--chromogenic ester incorporated into the strip toform a pyrrole compound, which in turn reacts with a diazonium salt toform a highly colored azo dye. The degree and intensity of the colortransition is proportional to the amount of leukocyte esterase or HLE inthe urine, which in turn, is proportional to the number of leukocytecells in the urine.

The reaction chemistry of the LEUKOSTIX® test strip is illustrated asfollows: ##STR3##

In particular, the LEUKOSTIX® assay for leukocyte esterase is based onthe splitting, i.e., hydrolysis, of the3-hydroxy-5-phenyl-pyrrole-N-tosyl-L-alanine ester (I) by an enzyme orenzymes to form 3-hydroxy-5-phenyl-pyrrole (II). The hydroxy-pyrrole(II) then reacts with a diazonium salt (III) (e.g.,1-diazo-2-naphthol-4-sulfonic acid) to produce azo dye (IV) having apurple color.

The reacted LEUKOSTIX® test strip is matched to a color chart havingfour color blocks of increasing color intensity from trace to 3+, whichin an average urine represents a leukocyte cell concentration of about10 to greater than 500 cells/μL (microliter), or 30 to greater than 1500ng/mL (nanograms per milliliter) of HLE. In low specific gravity testsamples, even smaller concentrations of HLE can be detected.

The intensity of the color is proportional to the amount of enzyme(e.g., HLE) present in the urine sample and, therefore, is directlyrelated to the number of leukocyte cells in the urine. An assay whichgenerates a color of 1+ or more is a definite indication that asignificant number of leukocyte cells are present in the urine sample.

The present day composition and method of assaying for leukocyte cellsis disclosed in Corey et al. U.S. Pat. No. 4,657,855 and Skjold et al.U.S. Pat. No. 4,637,979. As disclosed therein and as discussed above,the current LEUKOSTIX® test strips rely upon an amino acid ester, and inparticular an alanine ester, to provide a color transition in the assayfor leukocyte cells. Other patents related to assaying for an esteraseor protease enzyme include Hugl et al. U.S. Pat. Nos. 4,806,423 and4,814,271.

In contrast to present-day compounds and compositions used to assay forHLE and leukocyte cells, the method and test device of the presentinvention utilize a lactate ester, like a hydroxy-protected5-phenyl-3-hydroxy-pyrrolyl-L-lactate. The present lactate esters haveincreased reactivity relative to the corresponding alanine esters, suchas the N-tosyl alanine ester having structural formula (I) depictedabove. A combination of the present lactate esters and a diazonium saltcoupling agent also provide a more sensitive assay for HLE. The lactateesters, like the alanine esters, undergo a detectable or measurableresponse, like a color transition, upon contact with leukocyte cells,esterase or protease. The response is proportional to the concentrationof leukocyte cells, esterase or protease in a test sample.

Other patents and publications disclose the hydrolysis of indoxyl- andthioindoxyl-alanine esters to generate a color transition or otherdetectable response. These references include GB Patent No. 1,128,371;Janoff et al., Proc. Soc. Exper. Biol. Med., 136, pp. 1045-1049 (1971);Sweetman et al., Jour. Hist. Soc., 22, pp. 327-339; and Berger et al.U.S. Pat. No. 4,278,763.

To date, no known patent or publication discloses the use of a lactateester as an enzyme substrate in the assay for leukocyte cells, esteraseor protease. Dorn et al. U.S. Pat. No. 4,064,236 and Dorn et al., J.Med. Chem., 20, pp. 1464-1468 (1977) disclose inhibitors for pancreaticand granulocyte elastase. Some of the compounds contain a lactate moietyat the non-scissle and scissle points of cleavage. These compounds rangefrom being strong to very weak inhibitors. These compounds do notcontain activated leaving groups and are designed primarily asinhibitors to elastase with carbazate esters resembling alanine presentat non-hydrolytic sites to increase selectivity for inhibiting elastase.Similarly, Japanese Kokai JP 52/057,121 discloses lactoyl-polypeptidecompounds exhibiting potent antipepsin and anticathepsin inhibitoryactivity, wherein the lactate moiety again is incorporated in aninhibitory capacity. In contrast, the present lactate esters arechromogenic substrates having high esterase (HLE) enzymatic activity,and the ester linkage of the chromogenic lactate ester is the hydrolysissite.

J. Dufer et al., Ann. Pharm. Fr., 31, pp. 441-450 (1973) discloses a9-methoxyellipticine lactate salt having a decreased esterase activityin lymphocytes, and which was used against acute myeloblastic leukemia.The disclosed lactate salt is used as a solubilizing component for theellipticine, and has an inhibitory activity for esterase.

H. Moorlag et al., J. Org. Chem., 55, pp. 5878-5881 (1990) and H.Moorlag et al., Tetrah.; Assym., 2, pp. 705-720 (1991) disclose theenzymatic hydrolyses of a variety of α-substituted mandelic esters,α-substituted lactic esters and racemic α-substituted α-hydroxy estersusing pig liver esterase to determine enantioselectivity. Pig liveresterase showed no enantioselectivity for the α-substituted lacticesters.

F. Kraicsovits et al., Symp. Pap. IUPAC Int. Symp. Chem. Nat. Prod., 1,pp 37-40 (1978) discloses the effect of structure on the reactivity ofsubstrates in the presence of the serine protease, chymotrypsin, whereinparticular non-amino acid substrates contained the lactate moiety. Inthese examples, the lactate moiety is not at the hydrolysis site, butrather one residue removed from the hydrolysis site.

Publications such as J. W. Harper et al., Biochem., 23, pp. 2995-3002(1984); G. Digenis et al., J. Med. Chem., 29, pp. 1468-1476 (1986); A.Krantz et al., J. Med. Chem., 33, pp. 464-479 (1990); and D. W. Ingleset al., Biochem. J., 108, pp. 561-569 (1968) disclose that the naturalsubstrate at the HLE cleavage site is an amino acid, such as alanine orvaline.

In particular, the D. W. Ingles et al. publication discloses rates ofdeacylation of acyl-α-chymotrypsins wherein the nitrogen-hydrogenbonding capacity of the acylamino group of the substrate has beeneliminated by replacing the nitrogen with an oxygen (i.e., conversion toa lactate). When L-phenyl-alanyl was changed to L-phenyl-lactyl, thesubstitution resulted in a ten fold decrease in the enzymatic rate.Stereospecificity also decreased by a magnitude of up to 700 inreplacing the amino NH moiety with a lactate OR moiety, wherein R isacetate or carboxyphenyl.

Other publications disclosing the hydrolysis of lactates and/oralaninates include:

J. Suh et al., J. Am. Chem. Soc., 107, pp. 4530-4535 (1985);

J. Suh et al., J. Am. Chem. Soc., 98, pp. 1940-1947 (1976);

J. Sub et al., Biochemical and Biophysical Research Communications, 64,pp. 863-869 (1975);

L. C. Kuo et al., J. Mol. Biol., 163, pp. 63-105 (1983);

S. J. Hoffman et al., J. Am. Chem. Soc., 105, pp. 6971-6973 (1983);

P. L. Hall et al., J. Am. Chem. Soc., 91, pp. 485-461 (1968); and

M. W. Makinen et al., Proc. Natl. Acad. Sci. USA, 73, pp. 3882-3886(1976).

Therefore, in order to detect the onset and to monitor the progressionof a kidney or urogenital tract infection, an accurate and sensitiveassay.of urine for leukocyte cells, esterase or protease is needed forboth laboratory and home use. The assay should permit the detection andmeasurement of the leukocyte cells, esterase or protease in the testsample such that a correct diagnosis can be made and correct medicaltreatment implemented, monitored and maintained. In addition, it wouldbe advantageous if the assay method utilizes a dry phase test strip in adip-and-read format for the easy and economical determination ofleukocyte cells or HLE in urine.

Present day test strips for leukocyte cells need improvement in theareas of sensitivity and speed of assay. Therefore, it would be asignificant advance in the art of diagnostic assays if test strips forleukocyte cells were more sensitive to low concentrations of leukocytecells and provided assay results in about 60 seconds. It was towardsachieving these improvements that investigations resulting in thecomposition, device and method of the present invention were directed.

The method of the present invention provides a fast, accurate andtrustworthy assay for leukocyte cells, esterase or protease by utilizinga test strip having a test pad comprising a suitable carrier matrixincorporating a reagent composition of the present invention. Thereagent composition comprises a lactate ester, and in particular, ahydroxy-protected 5-phenyl-3-hydroxy-pyrrolyl-L-lactate. The compositionfurther comprises a buffer, and optionally can include an acceleratorcompound and/or a diazonium salt coupling agent. The reagent compositionis sensitive to trace concentrations of leukocyte cells, esterase orprotease. The present reagent composition enhances the sensitivity ofthe assay for leukocyte cells, esterase or protease, thereby providing amore accurate and trustworthy assay.

No known method of assaying urine or other test samples for leukocytecells, esterase or protease used a reagent composition comprising alactate ester of the present invention.

SUMMARY OF THE INVENTION

In brief, the present invention is directed to a new and improvedcomposition, test device and method of determining the presence orconcentration of a predetermined component in a test sample. The deviceincludes a test pad comprising a carrier matrix. The carrier matrixincorporates a reagent composition capable of interacting with apredetermined test sample component to produce a detectable response.For home use, the reagent composition produces a visually detectableresponse. For laboratory use, the reagent composition produces aresponse that is detectable visually or instrumentally. The carriermatrix of the test pad comprises a bibulous material, such as filterpaper; a nonbibulous material, such as a strip, layer or membrane of apolymerized material; or a combination thereof. A reagent composition ishomogeneously incorporated into the carrier matrix, and the carriermatrix holds the reagent composition homogeneously throughout thecarrier matrix while maintaining carrier matrix penetrability by thepredetermined component of the test sample.

More particularly, the present invention is directed to a method ofassaying urine for leukocyte cells, esterase or protease by utilizing anew and improved reagent composition. The reagent composition comprises:(a) a lactate ester capable of generating a detectable response uponinteraction with leukocyte esterase; and (b) a buffer.

The term "detectable response" as used herein means a change in, or anoccurrence of, a parameter in a test device. The detectable response iscapable of being perceived, either visually or instrumentally. Themagnitude of the detectable response is proportional to the presence andconcentration of a specific analyte in an aqueous test sample.Exemplary, but nonlimiting, detectable responses include a change in, oran occurrence of, color, fluorescence, reflectance, pH,chemilumnimescence, spectrophotometry or colorimetry.

In a preferred embodiment, the reagent composition comprises: (a) alactate ester having the structural formula (V) ##STR4## wherein A is analcohol blocking group; X is O, S, or NR² ; R is aryl or lower alkyl; R¹is hydrogen or lower alkyl; and R² is hydrogen, lower alkyl or aryl; and(b) a buffer.

With respect to stereochemistry, the lactate ester can be in the L-form,the D-form, or a racemic mixture of the D and L-forms. The L-form ispreferred. The composition optionally can include an acceleratorcompound, such as an alcohol having three to about 15 carbon atoms,and/or a diazonium salt coupling agent.

The method comprises contacting a test sample with the reagentcomposition, or a test device incorporating the reagent composition,then observing and measuring a detectable response, such as a colortransition, for the presence or concentration of leukocyte cells,esterase or protease.

In accordance with an important feature of the present invention, a moreaccurate and reliable assay for leukocyte cells, esterase or protease ina test sample is achieved because the reagent composition exhibits animproved sensitivity to the analytes of interest in comparison topresent-day compositions used to assay for leukocyte cells, esterase orprotease. Therefore, by utilizing the reagent composition of the presentinvention, which comprises a lactate ester and, optionally, a diazoniumsalt coupling agent, the assay for leukocyte cells, esterase orprotease, at trace through high concentrations, is more accurate.

Therefore, one aspect of the present invention is to provide a simple,accurate and reproducible method of assaying urine or other liquid testsamples for leukocyte cells, esterase or protease.

Another aspect of the present invention is to provide a method ofassaying urine for leukocyte cells, esterase or protease by utilizing areagent composition that provides increased sensitivity and accuracy inthe assay for HLE.

Another aspect of the present invention is to provide a new and improvedtest device for interaction with HLE in a test sample to produce avisible change in the test device, such as a change in color, which isindicative of the concentration of HLE in the test sample.

Yet another aspect of the present invention is to provide a sensitivemethod of detecting and assaying urine for HLE in a concentration rangeof about 5 to about 500 ng/mL, and correlating the assay to theconcentration of leukocyte cells (i.e., about 2 to about 175 cells/μL)in the urine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the method of the present invention, the assay ofurine for leukocyte cells, esterase, protease or HLE is accomplished byutilizing a reagent composition comprising a lactate ester, a buffer,and optionally, an accelerator compound and/or a diazonium salt couplingagent. By employing a reagent composition of the present invention, thelactate ester, such as the lactate ester of structural formula (V), ishydrolyzed by the leukocyte esterase (HLE) to provide, for example, thehydroxy-pyrrole compound of structural formula (II), which in turn caninteract with an optional diazonium salt to form an azo dye. The lactateesters of the present invention are readily hydrolyzed by HLE togenerate a measurable color transition or other detectable responsewithin about 60 to about 120 seconds.

The reagent composition of the present invention, which is capable ofdetecting HLE and therefore is capable of detecting leukocyte cells,comprises a lactate ester having the general structural formula (VI)##STR5## wherein A is an alcohol blocking group and B is a moietycapable of providing a detectable response, preferably a chromogenicresponse, when the lactate ester of structural formula (VI) ishydrolyzed to generate the compound B-OH. The lactate ester can be theD-form, the L-form or a racemic mixture of the D and L-forms. The L-formis preferred.

The moiety B-O- of the compound of structural formula (VI) is defined asthe residue of a compound B-OH. The moiety B-O- therefore can be theresidue of a substituted or unsubstituted pyrrole, thiophene or furan,for example. Other exemplary compounds having a residue B-O- include,but are not limited to, an azoresorcinol ester, a phenoxy ester (with anoxidative coupler), a leukoindophenol ester, an azo dye ester,5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3-aceticacid, a 2-substituted-6-hydroxy-benzothiazole derivative disclosed in WO90/00618 and EP 399 490 (both incorporated herein by reference) havingthe structure ##STR6## wherein: a) at least one of the carbons ofpositions 4, 5, 6 or 7 is linked to a chemical moiety containing ananion group which is attached to the benzene ring; and;

b) the carbon at position 2 is linked to a chemical moiety comprised ofat least two atoms which extend resonance of the benzothiazole ringsystem; and,

c) a nitrogen atom is located at position 3; and

d) a sulfur atom is located at position 1. A ω-nitrostyryl ester, aresorufin ester, an acridinone, a merocyanine, an8-hydroxy-2H-dibenz-(b,f)azepin-2-one, a dibenzo azepinone, adibenzothiazepinone, a coumarin ester, or a chemiluminescent compounddisclosed in Schaap U.S. Pat. Nos. 4,857,652 and 4,962,192, incorporatedherein by reference.

A preferred lactate ester has the general structural formula (V)##STR7## wherein A is an alcohol blocking group, X is O, S or NR², R isaryl or lower alkyl, R¹ is hydrogen or lower alkyl, and R² is hydrogen,lower alkyl or aryl. The lactate ester of general structural formula (V)is hydrolyzed by the enzyme leukocyte esterase or HLE to generate ahydroxy-compound, such as the hydroxypyrrole (II).

The lactate ester (V) is present in a reagent composition in aconcentration of about 0.5 to about 2 mM (millimolar), and preferablyabout 0.8 to about 1.5 mM. Within this concentration range, a sufficientamount of lactate ester is present in the reagent composition to providea sufficient color transition or other detectable response to detecttrace amounts of leukocyte cells.

The term "lower alkyl", as used herein, is an alkyl moiety containingone to about six carbon atoms. Exemplary, but nonlimiting, lower alkylgroups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,tert-butyl and all isomers of pentyl and hexyl. The lower alkyl groupcan be unsubstituted, or the lower alkyl group can be substituted,provided the substituent does not interfere with the ability of thecomposition or test device to detect leukocyte cells, esterase orprotease. Exemplary, but nonlimiting, substituents on the alkyl groupare alkoxy having one to six carbon atoms, halo, nitro, aryl, and amino.

The identity of the alcohol blocking group, i.e., A, of the lactateester of general structural formula (VI) is not particularly limited,and can be selected from essentially any blocking group typically usedto protect an alcohol moiety.

The alcohol blocking group A typically is the residue of a sulfonylchloride or a carboxylic acid chloride (i.e., an acyl chloride) and hasthe structural formula (VII) or (VIII) ##STR8## wherein R³ is an alkylgroup having three to about 22 carbon atoms, and preferably 3 to about 6carbon atoms, or R³ is an aryl group. When R³ is an alkyl group, thealkyl group can be functionalized, e.g., methoxysuccinyl.

As used herein, the term "aryl" with respect to R, R² and R³ means anyaromatic ring system. Nonlimiting examples of the term "aryl" include 5-and 6-membered aromatic ring systems like pyrrolyl, phenyl and pyridyl,as well as fused aromatic ring systems, like naphthyl. The aromatic ringsystem can be heterocyclic or carbocylic, and can be substituted orunsubstituted, provided that the substituent groups do not interferewith ability of the chromogenic lactate ester to hydrolyze in thepresence of leukocyte cells, esterase or protease. Exemplary, butnonlimiting, substituent groups are alkyl, halo, acyl, aryl, hydroxy,alkoxy, sulfuryl and amino. The aryl group preferably is a phenyl group,either unsubstituted or substituted with a relatively nonreactive group,such as a halo group or an alkyl or alkoxy group having one to about 10carbon atoms.

Exemplary, but nonlimiting, alcohol blocking groups are residues ofp-toluenesulfonyl chloride (tosyl chloride or TsCl), n-propylsulfonylchloride (n-PrSO₂ Cl), benzoyl chloride (PhCOCl), carbomethoxyethanesulfonyl chloride and thiophene sulfonyl chloride. Numerous otherspecific alcohol blocking groups are known to those skilled in the artand can be used as the A component of the present lactate esters. Forexample, numerous alcohol blocking groups are disclosed in T. W. Greeneet al., Protecting Groups in Organic Chemistry, 2Ed., (1991),incorporated herein by reference.

A preferred alcohol blocking group A has the structural formula (VII)and includes the sulfonyl moiety. To achieve the full advantage of thepresent invention, the alcohol blocking group of structural formula(VII) has a phenyl group as R³, wherein the phenyl group is substitutedwith a methyl or a methoxy moiety.

A preferred lactate ester of the present invention is a chromogeniccompound having the structural formula (IX): ##STR9## which is theL-form of a lactate ester of general structural formula (V), wherein Xis NR², R is phenyl and R¹ is hydrogen. In a more preferred embodiments,the chromogenic lactate ester has structural formula (X): ##STR10##wherein R² is hydrogen and A is p-toluenesulfonyl (i.e., Ts), i.e., theL-form of the lactate ester of structural formula (V), wherein X is NH,A is Ts, R is phenyl and R¹ is H.

Various lactate esters of structural formula (IX), wherein R² ishydrogen, have been prepared. These chromogenic lactate esters arelisted below in Table 1 as the 5-phenyl-3-hydroxy-pyrrole-(L)-lactateesters. The chromogenic lactate esters listed in Table 1 were preparedfrom the ethyl ester of lactic acid, wherein the hydroxy (i.e., alcoholgroup) of lactic acid has been blocked. The alcohol-blocked lactic acidethyl ester starting materials also are listed in Table 1. Each lactateester is in the L-form.

                                      TABLE 1                                     __________________________________________________________________________    Lactic Acid Ethyl     5-Phenyl-3-hydroxy-pyrrole                              Ester Analogs         Lactate Esters                                          __________________________________________________________________________    (L)-Ts--OCH(CH.sub.3)CO.sub.2 Et                                                                    Ts-(L)-Lac--OPP (X)                                     (L)-nPr--SO.sub.2 --OCH(CH.sub.3)CO.sub.2 Et                                                        nPr--SO.sub.2 -(L)-Lac--OPP (XI)                        (L)-PhCO--OCH(CH.sub.3)CO.sub.2 Et                                                                  PhCO-(L)-Lac--OPP (XII)                                 (L)-MeOSucc--SO.sub.2 --OCH(CH.sub.3)CO.sub.2 Et                                                    MeOSucc--SO.sub.2 -(L)-Lac--OPP (XIII)                  (L)-2-Thiophene-SO.sub.2 --OCH(CH.sub.3)CO.sub.2 Et                                                 Thiophene-SO.sub.2 -(L)-Lac--OPP(MV)                    __________________________________________________________________________     PPO = 5phenyl-3-hydroxy-pyrrole, Lac = lactate (--OCH(CH.sub.3)CO--), Ts      tosyl, PhCO = benzoyl, nPr = npropyl, MeOSucc = methoxysuccinyl (CH.sub.3     O(CO)CH.sub.2 CH.sub.2), and SO.sub.2 = sulfonyl                         

The synthesis of 3-(O-tosyl-(L)-lactoyl)-5-phenylpyrrole (IX) is typicalof the synthetic route used to prepare each lactate ester (X) through(XIV) listed in Table 1, or to prepare other lactate esters having thegeneral structural formula (V) or (VI).

The following examples are provided to demonstrate how to make and usethe lactate esters, compositions and test devices of the presentinvention. Various preferred embodiments are described in experimentaldetail hereafter. However, the following examples are illustrative only,and are not intended as limiting the scope of the invention disclosedand claimed herein.

In the examples and throughout the specification, the followingabbreviations have been used:

    ______________________________________                                        mg =      milligram                                                           g =       gram                                                                kg =      kilogram                                                            cm =      centimeter                                                          L =       liter                                                               mL =      milliliter                                                          M =       molar                                                               mM =      millimolar                                                          mol =     gram molecular formula (moles)                                      mmol =    gram molecular formula × 10.sup.-3 (millimoles)               aq =      aqueous                                                             hr =      hour                                                                ______________________________________                                    

Infrared (IR) spectra were obtained with a Perkin-Elmer Model 710B or237 infrared spectrophotometer as solutions in CDCl₃ unless otherwisenoted; the 1602 cm⁻¹ band of polystyrene film was used as an externalcalibration standard. Signals are reported as cm⁻¹.

Proton magnetic resonance (¹ H NMR) spectra were obtained at 300 MHzusing a GE GN300NB spectrometer or at 60 MHz using a Varian T-60spectrometer; spectra were obtained in CDCl₃ solution unless otherwisenoted. Chemical shifts are reported in parts per million (ppm) downfieldfrom an internal standard (i.e., tetramethylsilane).

Carbon-13 magnetic resonance (¹³ C NMR) spectra and DEPT magneticresonance spectra also were obtained using the GE GN300NB spectrometerwith Fourier transform and with full proton broad-band noise decoupling;spectra were obtained in acetone-d₆, CD₃ OD, DMSO-d₆ or CDCl₃ solutionunless otherwise noted. Carbon shifts are reported in parts per milliondownfield from tetramethylsilane.

Mass spectra (MS) were obtained on a Hewlett-Packard 5985A spectrometeroperating in either chemical ionization (CI), electron impact (EI) orfast atom bombardment (FAB) mode. High-resolution mass spectra wereobtained on an AEI MS-902 spectrometer. Additional spectra were obtainedfrom the Michigan State University Mass Spectroscopy Facility, EastLansing, Mich. 48824.

Optical rotations were obtained on a Model 141 Polarimeter, availablefrom Perkin-Elmer Corporation.

The chromogenic lactate esters (X)-(XIV) were prepared to illustratesynthesis of the lactate esters of the present invention. While theseexamples disclose specific starting materials and lactate esters, it isenvisioned that the synthetic procedures are applicable to a broad rangeof species included within the generic class of lactate esters ofstructural formula (VI).

EXAMPLE 1 Synthesis of the Lactate Ester of Structural Formula X##STR11##

Tosyl chloride (TsCl) (8.1 g, 42.3 mmol) was added with stirring to asolution of the ethyl ester of (L)-lactic acid (5.0 g, 4.81 mL, 42.3mmol) in methylene chloride (CH₂ Cl₂) (50 mL), which was chilled to 0°C. (ice-bath) under an argon blanket. Triethylamine (TEA) (5.57 g, 7.67mL, 55.0 mmol) was then added, dropwise, to the resulting solution. Theresulting reaction mixture then was stirred for seven hours at 0° C. Thereaction mixture next was poured over a solution of 1M aqueoushydrochloric acid (aq HCl) (75 mL) and ice (75 g). The CH₂ Cl₂ organiclayer was separated from the HCl layer, then the organic layer was driedover magnesium sulfate (MgSO₄). After filtering, the organic layer wasconcentrated in vacuo to yield 10.5 g (91%) of crude (L)-tosyl lactateethyl ester.

The crude (L)-tosyl lactate ethyl ester (10.5 g, 38.6 mmol) wasdissolved in absolute ethanol (12 mL), and the resulting solution wasadded dropwise via an addition funnel to a chilled (0° C. ice bath)solution of sodium hydroxide (NaOH) (1.8 g, 46 mmol) in distilled water(20 mL) over a fifteen minute time period. After stirring the resultingreaction mixture for five hours under an argon blanket at 0° C., thereaction was carefully quenched by the dropwise addition of concentratedHCl to lower the pH of the reaction mixture to pH 2. Solid sodiumchloride (NaCl) then was added to saturate the reaction mixture, and theaqueous layer was extracted four times with 50 mL portions of CH₂ Cl₂.The combined portions of CH₂ Cl₂ were dried over MgSO₄, filtered, andconcentrated in vacuo to provide 7.17 g (76%) of crude(L)-O-tosyl-lactic acid (XIV).

Under an argon blanket, the crude (L)-O-tosyl-lactic acid (XIV) (1.5 g,6.15 mmol) was placed in a 25 mL round-.bottomed flask equipped with areflux condenser. Thionyl chloride (SOCl₂) (10.12 g, 6.2 mL, 85 mmol)was added to the flask, then the flask was placed into a preheated (50°C.) oil bath. The contents of the flask were stirred for two hours at50° C. The flask then was cooled to room temperature, and finally placedin an ice bath. Next, ice cold hexane (25 mL) was added to the flask. Nosolid product was observed, therefore the solution in the flask wasconcentrated in vacuo to yield 1.6 g (quant.) of a crude yellow oil,i.e., (L)-O-tosyl-lactic acid chloride.

In a separate flask, pyridine (1.5 mL, 18.6 mmol) was added to a chilledsolution (0° C. ice bath) of 5-phenyl-3-hydroxy pyrrole (PPO-H) (986 mg,6.2 mmol) in CH₂ Cl₂ (30 mL) under an argon blanket, rapidly anddropwise, followed immediately by a rapid dropwise addition of asolution of (L)-O-tosyl-lactic acid chloride (1.6 g, 6.11 mmol) in CH₂Cl₂ (5 ml). The residual contents then were added dropwise with anadditional 5 ml of CH₂ Cl₂. The resulting reaction mixture was stirredfor one hour at 0° C., then allowed to warm to room temperature over anapproximately one hour time period. The reaction mixture then wasstirred overnight at room temperature (16 hr).

Next, the reaction mixture was extracted two times with 25 mL portionsof aq. 1M HCl. The combined portions of HCl then were back-extractedwith CH₂ Cl₂ (25 ml). The CH₂ Cl₂ extractant finally was extracted twotimes with 25 mL portions of saturated aqueous sodium bicarbonate. Afteragain back-extracting the aqueous phase with CH₂ Cl₂, the combinedportions of CH₂ Cl₂ were treated with norite carbon and MgSO₄, thenfiltered, and finally concentrated in vacuo.

The resulting oil was dissolved in hexane/ethyl acetate (1:1, 25 mL).The resulting solution was treated with norite carbon, then filtered,and finally concentrated in vacuo to yield 1.7 g of solid crude lactateester (X). The crude lactate ester (X) was triturated with warm hexane(10 mL), then was triturated two times with 8 mL portions ofhexane/ethyl acetate (4:1, 2×8 mL). After the organic solvents weredecanted, the solid product was dried under vacuum to provide 1.04 g ofa pink solid. The pink solid was dissolved in hexane/ethyl acetate(1:1), treated with norite carbon, filtered, and concentrated in vacuoat room temperature to yield 932 mg of lactate ester (X) as a cremecolored solid.

The lactate ester of structural formula (X) was analyzed by proton (¹ H)and carbon-13 (¹³ C) nuclear magnetic resonance, elemental analysis(C/H/N), mass spectroscopy (MS), infrared spectroscopy (IR) and opticalrotation. The analytical data is summarized below and confirms thestructure of lactate ester (X).

¹ H NMR (CDCl₃, ppm): 1.65 (d, 3H), 2.42 (s, 3H), 5.12 (qu., 1H), 6.26(dd, 1H), 6.84 (dd, 1H), 7.18-7.5 (m, 7H), 7.84 (d, 2H), 8.15 (brs, 1H).

¹³ C NMR (CDCl₃, ppm): 18.51 (methyl), 21.66 (methyl), 73.96 (CH,lactate) , 93.39 (CH, pyrrole) , 107.89 (CH, pyrrole) , 123.84 (CH,arom.), 126.85 (CH, arom.), 128.12 (CH, arom.), 128.97 (CH, arom.),129.87 (CH, arom.), 166.68 (C=O) .

C/H/N: calc.: C: 62.39, H: 4.97, N: 3.63, S: 8.33; found: C: 62.44, H:5.03, N: 3.57, S: 8.65.

EI/MS (18 EV, DIP): 385 (M+, 21.8), 227 (2.3), 199 (22.4), 158 (67.5),155 (BASE), 91 (41.7).

IR: (CDCl₃, cm⁻¹): 3450, 3022, 1770, 1600, 1570, 1550, 1514, 1450, 1370,1260, 1240, 1180, 1080, 1020, 980.

Optical rotation (λ=578) on a 10 mg sample in 1 mL methanol (MeOH) atroom temperature in a 1 cm cell: (-64.8°) .

EXAMPLES 2-5 Synthesis of the Lactate Esters of Structural Formulae(XI)-(XIV)

The 5-phenyl-3-hydroxy-pyrrole lactate esters of structural formulae(XI)-(XIV) listed in Table 1 were prepared in an essentially identicalmanner as the chromogenic lactate ester (X) of Example 1. However,different alcohol blocking groups were used, i.e., n-propylsulfonyl,benzoyl, methoxysuccinyl sulfonyl and thiophene sulfonyl, in thesynthesis chromogenic of lactate esters (XI)-(XIV), respectively.

The preparation of the lactate esters of structural formulae (XI)-(XIV)was confirmed by the analytical data summarized below.

nPr-SO₂ -(L)-Lac-OPP (XI)

¹ H NMR (CDCl₃, ppm): 1.10 (t, 3H), 1.75 (d, 3H), 2.20 (m, 2H), 3.30 (m,2H), 5.35 (qu., 1H), 6.41 (dd, 1H), 6.95 (dd, 1H), 7.20-7.60 (m, 5H),8.30 (brs, 1H).

¹³ C NMR (CDCl₃, ppm): 12.87 (methyl), 17.23 (methylene), 18.59(methyl), 53.64 (methylene), 73.49 (CH, lactate) , 98.32 (CH, pyrrole),107.94 (CH, pyrrole), 123.86 (CH, arom.), 126.90 (CH, arom.), 128.96(CH, arom.) , 166.5 (C=O).

C/H/N: calc.: C: 56.97, H: 5.64, N: 4.15, S: 9.49; found: C: 57.06, H:5.75, N: 3.87, S: 9.91.

FAB/MS: 337.2 (M+, 55), 159 (BASE).

IR: (CDCl₃, cm⁻¹): 3400, 3010, 2980, 1768, 1573, 1512, 1454, 1400, 1360,1254, 1169, 1116, 1084, 984.

Optical rotation (λ=578) on a 13 mg sample in 1 mL MeOH at roomtemperature in a 1 cm cell: (-48.5°).

PhCO-(L)-Lac-OPP (XII)

¹ H NMR (CDCl₃, ppm): 1.75 (d, 3H), 5.55 (qu., 1H), 6.40 (dd, 1H), 6.95(dd, 1H), 7.15-7.7 (m, 8H), 8.1 (d, 2H), 8.2 (brs, 1H).

¹³ C NMR (CDCl₃, ppm): 17.13 (methyl), 69.08 (CH, lactate), 98.57 (CH,pyrrole), 108.03 (CH, pyrrole), 123.82 (CH, arom.), 126.68 (CH, arom.),128.41 (CH, arom.), 128.89 (CH, arom.), 129.91 (CH, arom.), 133.33 (CH,arom.), 165.98 (C=O), 166.48 (C=O).

C/H/N: calc.: C: 71.64, H: 5.07, N: 4.18; found: C: 71.92, H: 5.22, N:4.28.

EI/MS (18 EV, DIP): 333 (M+, 3.1), 177 (42.8), 149 (22.2), 105 (BASE),77 (0.7).

IR: (CDCl₃, cm⁻¹): 3400, 3000, 1763, 1723, 1606, 1585, 1573, 1512, 1452,1318, 1177, 1113.

Optical rotation (λ=578) on a 12 mg sample in 1 mL MeOH at roomtemperature in a 1 cm cell: (+13.3°).

MeOSucc-SO₂ -(L)-Lac-OPP (XIII)

¹ H NMR (CDCL₃, ppm): 1.78 ⁻ (d, 3H), 1.98 (t, 2H), 825 2.75 (t, 2H),2.75 (s, 3H), 5.37 (qu., 1H), 6.42 (brs, 1H), 6.98 (brs, 1H), 7.2-7.55(m, 5H), 8.2 (brs, 1H).

¹³ C NMR (CDCl₃, ppm): 18.52 (methyl), 28.42 (methylene), 47.29(methylene), 52.4 (methyl), 74.14 (CH, lactate), 98.37 (CH, pyrrole),107.93 (CH, pyrrole), 123.89 (CH, arom.), 126.94 (CH, arom.), 128.98(CH, arom.) .

C/H/N: calc.: C: 53.54, H: 4.99, N: 3.67, S: 8.39; found: C: 53.05, H:5.07, N: 3.41, S: 7.75.

FAB/MS: 381 (M+, 63), 186 (8.5), 159 (BASE), 91 (12), 55 (24.5).

IR: (CDCl₃, cm⁻¹): 3450, 3000, 1751, 1741, 1572, 1513, 1453, 1439, 1359,1319, 1255, 1208, 1030, 984.

Optical rotation) (λ=578) on a 7 mg sample in 1 mL MeOH at roomtemperature in a 1 cm cell: (-27.1°).

2-Thiophene-SO₂ -(L)-Lac-OPP (XIV)

¹ H NMR (CDCl₃, ppm): 1.70 (d, 3H), 5.19 (qu., 1H), 6.32 (dd, 1H), 6.85(dd, 1H), 7.12 (t, 1H), 7.20-7.50 (m, 5H), 7.70 (dd, 1H), 7.79 (dd, 1H),8.19 (brs, 1H).

¹³ C NMR (CDCl₃, ppm): 18.44 (methyl), 74.91 (CH, lactate), 98.34 (CH,pyrrole), 107.90 (CH, pyrrole), 123.85 (CH, arom.), 126.85 (CH, arom.),127.54 (CH, thiophene), 128.95 (CH, arom.), 134.09 (CH, thiophene),134.71 (CH, thiophene).

C/H/N: calc.: C: 54.1, H: 3.98, N: 3.71, S: 16.98; found: C: 53.4, H:4.16, N: 3.69, S: 16.81.

EI/MS (18 EV, DIP): 377 (M+, 63.1), 236 (4.4), 191 (13.8), 164 (4.9),159 (BASE), 147 (66.1), 99 (8.2), 83 (1.1).

IR: (CDCl₃, cm⁻¹): 3450, 3050, 2940, 1760, 1573, 1509, 1453, 1404, 1378,1084, 1018.

Optical rotation) (λ=578) on a 12 mg sample in 1 mL MeOH at roomtemperature in a 1 cm cell: (-34.3°).

Composition and Test Device

In addition to a lactate ester having a general structural formula (VI),the reagent composition includes a buffer. The buffer is a compoundwhich, when contacted with an aqueous test sample, provides a suitablepH for the reaction. Preferably, the buffer is capable of producing a pHin the range of about 7 to about 10 and, optimally, about 8.5 to about9.0.

A buffer is included in the reagent composition of the present inventionin a concentration of about 200 to about 600 mM, although in particularsituations the concentration of the buffer can be above or below thisrange.

Therefore, a reagent composition of the present invention is buffered toa suitable pH with a buffer such as carbonic acid; BICINE; CHES; borate;phosphate; 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol;3,3-dimethylglutaric acid; 3-N-morpholinopropanesulfonic acid (MOPS);1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-TRIS); tri(hydroxymethyl)aminomethane (TRIS);tris(hydroxymethyl)aminomethane-maleic acid (TRIS-maleate);tris(hydroxymethyl)aminomethane-malonic acid (TRIS-malonate);3-N-(trishydroxymethyl)methylamino-2-hydroxypropanesulfonic acid(TAPSO); 2-([tris(hydroxymethyl)methyl]amino)ethanesulfonic acid (TES);N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); and otherbuffers well known in the art, or combinations thereof. A preferredbuffer is boric acid-NaOH.

A composition of the present invention therefore comprises a lactateester having the structural formula (VI) and a buffer. As previously.discussed, the specific identity of the lactate ester having thestructural formula (VI) and of the buffer is not particularly limited.However, preferred lactate esters, like a lactate ester of structuralformula (V), and buffers produce optimized assay results, i.e., adetectable and differentiable response in a short time. Assayoptimization can be promoted further by including an acceleratorcompound and/or a diazonium salt coupling agent in the reagentcomposition.

An "accelerator compound" is any compound that increases the rate ofhydrolysis of a lactate ester of structural formula (V) or (VI) byleukocyte cells, esterase or protease. An accelerator compound isselected from such chemically diverse classes of substances as alcohols;and pyridine, imidazole and derivatives thereof. These acceleratorcompounds are disclosed in Berger et al. U.S. Pat. No. 4,299,917,incorporated herein by reference. Hydrophobic alcohols are especiallyuseful accelerator compounds to increase leukocyte activity. Branchedalcohols also have been shown to be useful accelerators of esteraseactivity. To achieve the full advantage of the present invention,decanol is utilized as the accelerator compound. An accelerator compoundis present in the reagent composition in an amount of 0 to about 4%(v/v), and preferably in about 0.1% to about 2% (v/v), of thecomposition. To achieve the full advantage of the present invention, theaccelerator compound is present in an amount of about 1% to about 2%(v/v) of the composition.

The present reagent composition also can include a diazonium salt as acoupling agent. The diazonium salt is present at a concentration of 0 toabout 2 mM, and preferably 0.1 to about 1 mM. To achieve the fulladvantage of the present invention, the diazonium salt coupling agent ispresent at a concentration of about 0.5 to about 1 mM. The diazoniumsalt interacts with a hydroxy compound B-OH, such as the hydroxy-pyrrole(II), which is generated by hydrolysis of a lactate ester of structuralformula (VI) by leukocyte cells, esterase or protease. The hydroxycompound then interacts with the diazonium salt to yield an azo dyewhich exhibits a deep, distinctive color, which is indicative of thepresence of leukocyte cells, esterase or protease.

Often, the hydroxy compound, such as the hydroxy-pyrrole (II), itself isa distinct ultraviolet (UV) absorbing compound and therefore also canserve as an indicator to detect the presence or concentration ofleukocyte cells, esterase or protease in a test sample. The degree andintensity of the color change of the reagent composition increases asthe concentration of hydroxy-pyrrole (II) increases. The concentrationof hydroxy-pyrrole (II) is directly proportional to the amount ofleukocyte cells, esterase or elastase in the test sample. Therefore, theincrease in absorbance generated by production of hydroxy-pyrrole (II)in the hydrolysis of chromogenic lactate ester (V) (or by production ofthe hydroxy compound B-OH in the hydrolysis of chromogenic lactate ester(VI)) can be correlated to the amount of leukocyte cells, esterase orelastase in the test sample. However, a more spectacular blue or redcolor transition results when a diazonium salt is present to interactwith a hydroxy compound B-OH or the hydroxy-pyrrole (II) to form an azodye. The more spectacular color change provides a more differentiablecolor change, and accordingly, a more accurate assay.

The diazonium salt coupling agent has a general structural formula

    ARN.sup.+ .tbd.N,

wherein Ar is an aryl group. More particularly, the diazonium salttypically is an aromatic diazonium salt having the general structuralformula: ##STR12## wherein R⁴, either the same or different, ishydrogen, lower alkyl or aryl, or wherein two adjacent R⁴ groupstogether form a fused ring system, with the proviso that one of R⁴ is-N⁺ .tbd.N, i.e., diazonium; Y is N or CR⁵, wherein R⁵ is hydrogen orlower alkyl; and D⁻ is an anion, such as chloride, bromide or othersuitable counterion for the diazonium moiety.

The term "fused ring system", as used herein, means two or more aromaticrings that share a pair of carbon atoms. For example, in the diazoniumsalt having structural formula (XVII), ##STR13## both G groups togethercan form a fused ring system, wherein both G groups together constitute-(CH-)₄, e.g., the diazonium salt of structural formula (XVIII). The Fgroup is hydrogen, lower alkyl or hydroxy. ##STR14## Another example ofa fused ring system is the composition of structural formula (XIX) ,##STR15## wherein both G groups together constitute -(CH=CH-CH=N)-.Therefore, a fused ring system is polynuclear, aromatic, andheterocyclic or homocyclic.

A diazonium salt which is zwitterionic is a preferred coupling agent.The zwitterionic diazonium compound is a species of diazonium saltwherein the counterion (i.e., the anion) of the diazonium moiety iscovalently bound to the ring system. Examples of such anions include,but are not limited to, sulfonate (SO₃ ⁻), carbonate (CO₂ ⁻) andphosphonate (PO₃ ⁻). Zwitterionic diazonium salts have the generalstructural formula (XVII), illustrated above, wherein F is hydrogen,lower alkyl or hydroxy; D is a covalently bound anion; G, either thesame or different, is hydrogen, lower alkyl or aryl, or both G groupstogether form a fused ring system.

Various diazonium salts are disclosed in Skjold et al. U.S. Pat. No.4,637,979; Hugl et al. U.S. Pat. No. 4,806,423; and Hugl et al. U.S.Pat. No. 4,814,271, incorporated herein by reference. Specific,nonlimiting examples diazonium salts useful in the composition andmethod of the present invention are 1-diazo-2-naphthol-4-sulfonate and1-diazophenyl-3-carbonate. Other nonlimiting examples of diazonium saltsare 4-diazo-3-hydroxy-1-naphthylsulfonate (DNSA),4-diazo-3-hydroxy-7-nitro-1-naphthylsulfonate (NDNSA),4-diazo-3-hydroxy-1,7-naphthyldisulfonate,2-methoxy-4-(N-morpholinyl)benzene diazonium chloride,4-diazo-3-hydroxy-7-bromo-l-naphthylsulfonate,4-diazo-3-hydroxy-7-cyano-1-naphthylsulfonate, and4-diazo-3-hydroxy-7-[1-oxopropyl]-1-naphthylsulfonate, illusrated belowby structural formulae (XX)-(XXVI), respectively. ##STR16## A preferreddiazonium salt is the compound of structural formula (XXI), whichenhances the color transition, and accordingly the sensitivity, of theassay for leukocyte cells, esterase or proteaseo

Therefore, the reagent composition of the present invention, comprisinga lactate ester of structural formula (VI), a buffer, and, optionally,an accelerator compound and/or a diazonium salt coupling agent, isutilized in an improved method to determine the presence or theconcentration of leukocyte cells, esterase or protease, or HLE, inliquid test samples. The reagent composition interacts with HLE tohydrolyze the lactate ester of structural formula (VI), which generatesa differentiable and measurable response, such as a color transition.The response can be detected visually or by instrument. Furthermore, inaddition to the ingredients described above, the reagent composition ofthe present invention also can include a sufficient amount of variousother optional ingredients.

Optional ingredients that do not materially alter the nature or thefunction of the essential ingredients, and that do not interfere withthe assay for leukocyte cells, esterase or protease also can be includedin the reagent composition. For example, the reagent compositionoptionally can include a compound to improve wetting of the test pad ofthe test device by the liquid sample. This compound typically is ananionic surfactant or a nonionic surfactant. An anionic surfactant, suchas long carbon chain sulfates or sulfonates, like pentyl to dodecylsulfates or sulfonates, dioctyl sodium sulfosuccinate and sodiumdodecylbenzene sulphonate, are the preferred surfactants. The surfactantis included in the reagent composition in a concentration of 0% to about0.4%, and preferably in a concentration of about 0.05% to about 0.2%, bytotal weight of the reagent composition.

The reagent composition also can include a polymeric material foruniformity of the color transition of the test device. Polymericmaterials can include, but are not limited to, polyvinylpyrrolidone,polyvinyl alcohol, gum arabic, gelatin, algin, carrageenan, casein,albumin, methyl cellulose and similar natural and synthetic polymericmaterials. The preferred polymeric material is a polyvinylpyrrolidone ofaverage molecular weight about 10,000 to about 200,000 and availablecommercially from ISP Corp., New York, N.Y. The polymeric materialgenerally is included in the reagent composition in an amount of 0% toabout 4%, and preferably from about 0.5% to about 2%, by total weight ofthe reagent composition.

The carrier for the ingredients included in the reagent compositioncomprises water. However, because of the limited water solubility ofparticular ingredients, organic solvents such as methanol, ethanol,isopropyl alcohol, acetone, dimethylformamide, dimethylsulfoxide, andsimilar solvents can be included in the carrier. The selection of asuitable organic solvent or solvents, in addition to water, to includein the carrier of the reagent composition is within the capability ofthose skilled in the art of designing diagnostic assays.

As previously described, the reagent composition undergoes a response,and preferably a color transition, upon contact with a test sample toassay for the presence or concentration of leukocyte cells, esterase orprotease. The intensity and degree of the color transition are used toquantitatively determine the concentration of leukocyte cells in thetest sample. In accordance with an important feature of the presentinvention, a reagent composition of the present invention provides asufficiently resolved and differentiated color transition such that theconcentration of leukocyte cells in a test sample can be measured andaccurately determined without the use of color-measuring instruments,such as spectrophotometers or colorimeters. However, if desired, suchcolor-measuring instruments can be used to measure the difference incolor degree and intensity between the test sample and a solution havinga known concentration of leukocyte cells, esterase or protease.

To demonstrate the method of the present invention, a reagentcomposition comprising a lactate ester of general structural formula (V)and a buffer was used in a dry phase test strip assay for HLE. Thereagent composition also can be used in a wet phase assay for HLE. Thedry phase or the wet phase assay for HLE can be correlated to theleukocyte cell concentration of the test sample.

A dry phase test strip assay utilizing the present reagent compositionis performed in accordance with methods well known in the art. Ingeneral, the assay for HLE is performed by contacting the urine or othertest sample with an analyte detection device that incorporates thereagent composition. The analyte detection device can be dipped into thetest sample, or the test sample can be applied to the analyte detectiondevice dropwise. The resulting change in color of the analyte detectiondevice demonstrates the presence of HLE; and, if so designed, theresulting color transition can be compared to a standardized color chartto provide a quantitative measurement of the concentration of HLE, andtherefore leukocyte cells, in the test sample.

Typically, the analyte detection device is a reagent-impregnated teststrip, designed either as a single pad test strip (to assay only for asingle analyte) or as a multiple pad test strip (to assay for severalanalytes simultaneously). For either type of reagent impregnated teststrip, the test strip includes a support strip, or handle, normallyconstructed from a hydrophobic plastic, and a reagent test pad,comprising a bibulous or a non-bibulous carrier matrix incorporating thereagent composition. In general, the carrier matrix is an absorbentmaterial that allows the test sample to move, in response to capillaryforces, through the carrier matrix to contact the reagent compositionand produce a detectable or measurable color transition.

The carrier matrix can be any substance capable of incorporating thechemical reagents required to perform the assay of interest, as long asthe carrier matrix is substantially inert with respect to the chemicalreagents, and is porous or absorbent relative to the soluble componentsof the liquid test sample. The expression "carrier matrix" refers toeither bibulous or nonbibulous matrices that are insoluble in water andother physiological fluids. Suitable bibulous matrices include filterpaper, sponge materials, cellulose, wood, woven and nonwoven fabrics andthe like. Nonbibulous matrices include glass fiber, polymeric films, andpreformed or microporous membranes. Other suitable carrier matricesinclude hydrophilic inorganic powders, such as silica gel, alumina,diatomaceous earth and the like; argillaceous substances; cloth;hydrophilic natural polymeric materials, particularly cellulosematerial, like cellulosic beads, and especially fiber-containing paperssuch as filter paper of chromatographic paper; synthetic or modifiednaturally-occurring polymers, such as crosslinked gelatin, celluloseacetate, polyvinyl chloride, polyacrylamide, cellulose, polyvinylalcohol, polysulfones, polyesters, polyacrylates, polyurethanes,crosslinked dextran, agarose, and other such crosslinked andnoncrosslinked water-insoluble hydrophilic polymers. Hydrophobic andnonabsorptive substances are not suitable for use as the carrier matrixof the present invention. The carrier matrix can be of differentchemical compositions or a mixture of chemical compositions. The matrixalso can vary in regards to smoothness and roughness combined withhardness and softness. However, in every instance, the carrier matrixcomprises a hydrophilic or absorptive material. The carrier matrix ismost advantageously constructed from bibulous filter paper ornonbibulous polymeric films. The handle usually is formed from ahydrophobic material such as cellulose acetate, polyethyleneterephthalate, polycarbonate or polystyrene.

If the test strip is designed to assay HLE in a test sample, the carriermatrix can be any bibulous or nonbibulous material that allows thesoluble components of the test sample to permeate and saturate the testpad of the test strip that is impregnated with the reagent composition.A preferred carrier matrix is a hydrophilic, bibulous matrix, includingcellulosic materials, such as paper, and preferably filter paper. Thecarrier matrix also can be a hydrophilic, nonbibulous matrix, includingpolymeric films, such as a polyurethane or a crosslinked gelatin. Suchcarrier matrices possess all of the qualities required of a carriermatrix of the present invention, such as suspending and positioning theingredients included in the reagent composition, and permeability of thesoluble components of the test sample through the carrier matrix.

To achieve the full advantage of the present invention, the reagentcomposition is impregnated into a suitable carrier matrix and utilizedin a dry phase test strip for the assay of leukocyte cells, esterase orprotease in a test sample. The method of the present invention providesan economical, accurate and reliable assay, that can be performed athome or in the laboratory, for the presence or concentration of HLE, andtherefore leukocyte cells, in a test sample. In addition, the method ofthe present invention allows detection, differentiation and measurementof a trace amount of leukocyte cells in the test sample, thereforemaking the assay more useful clinically.

In accordance with the method of the present invention, a test devicewas prepared which was sensitive to the presence of leukocyte esterase.An aqueous solution including 0.8M borate-sodium hydroxide buffer (pH8.8) and 1% by weight of polyvinylpyrrolidone (PVP K-60 available fromISP Corp., Wayne, N.J.) first was prepared. A 4 inch wide strip ofWHATMAN 3MM filter paper, available commercially from Whatman, Ltd.,Maidenhead, Kent, U.K., then was impregnated with the aqueous solution.The impregnated filter paper strip next was dried in an Air Foil paperdryer, available from Thermoelectron, Kaukauna, Wis., at 79° C. to 121°C. for about 5 to 6 minutes.

The dried, impregnated filter paper strip then was impregnated a secondtime by immersion into an acetone solution containing 1.1 mM of alactate ester of structural formula (IX)-(XIII), 0.7 mM4-diazo-3-hydroxy-7-nitro-1-naphthylsulfonate (i.e., the compound ofstructural formula (XX), also named 6-nitro-1,2-naphthoquinone diazide),1.5% (v/v) 1-decanol and 3% (v/v) dimethylsulfoxide. After the secondimpregnation, the filter paper strip was dried at 60° C. for about 5 to6 minutes to provide an off-white filter paper strip. The dried,twice-impregnated filter paper pad is secured to an opaque ortransparent hydrophobic plastic handle with double-sided adhesive tape.The dried and twice-impregnated filter paper strip was next cut to anappropriate size for a test strip assay, such as a pad having dimensionsof about 0.2 in (inch) (0.5 cm) by about 0.2 in (0.5 cm).

It should be understood that it is well within the experimentaltechniques of those skilled in the art of preparing test devices todetermine the proper balance between size of reagent pad, the strengthof reagent composition solutions, the amount of test sample, and themethod of introducing the test sample to the test strip, such as bypipetting rather than dipping, in order to design a quantitative assayfor leukocyte cells, esterase or protease utilizing the method andcomposition the present invention.

In addition, it should be understood that the carrier matrix can besaturated or impregnated by immersing the carrier matrix into a singleaqueous, or aqueous-organic solvent, solution including all of theessential and optional ingredients of the reagent composition. However,the two step method utilizing two immersions is preferred becauseparticular reagent composition ingredients have relatively low watersolubilities.

To perform an assay for leukocyte cells, esterase or protease, theresulting test strip is contacted with a urine sample for a sufficienttime to saturate the test pad with the sample. After waiting apredetermined time, such as from about one to about two minutes, thetest strip is examined, either visually or by instrument, for aresponse. A color transition in the test pad reveals the presence orconcentration of leukocyte cells, esterase or protease in the testsample.

In many cases, simple visual observation of the test strip provides thedesired information. If more accurate information is required, a colorchart bearing color spots corresponding to various known concentrationsof leukocyte cells can be prepared for the particular reagentcomposition used in the test strip. The resulting color of the teststrip after contact with the test sample then can be compared with thecolor spots on the chart to determine the concentration of leukocytecells in the test sample. In addition, the dry phase test strip assaycan be made more accurate by employing instrumental spectrophotometricor colorimetric techniques, as opposed to visual techniques, to measurethe degree and intensity of the color transition, especially at trace tolow concentrations.

To demonstrate the new and unexpected results achieved by the method ofthe present invention, dry phase test strips incorporating a reagentcomposition of the present invention were used to assay standardizedsolutions including HLE. Individual test strips each incorporating onechromogenic lactate ester having a structural formula (X) through (XIV)and a diazonium salt coupling agent were quickly immersed intostandardized solutions containing 0, 19 or 48 ng/mL HLE, then removed.Approximately two minutes after contacting a standardized HLE solution,the reflectance of the test pad of the test strip was measured at 557 nm(nanometers) on a CLINITEK® 10 clinical reflectance meter, availablefrom Miles, Inc., Elkhart, Ind.

The reflectance, R, as taken from the reflectance scale of zero to one,was incorporated into the Kubelka-Munk function:

    K/S=(1-R.sup.2 /2R),

wherein K is the absorption coefficient, S is the scattering coefficientand R is reflectance. The reflectance values determined at 557 nm wereused to calculate the K/S values. The K/S values are proportional totest strip color, therefore the greater the K/S value, the greater thedegree and intensity of the color transition of the test strip and thegreater the concentration of HLE in the test strip.

Test strips, each incorporating a different chromogenic lactate esterhaving a structural formula (X) through (XIV), were compared to thecorresponding amino acid ester, i.e., the alanine ester, for an abilityto assay for HLE. For example, the L-form of the lactate ester ofstructural formula (X) has the structure ##STR17## Lactate ester (X) wascompared to the corresponding alanine compound (I) for an ability toassay urine for HLE, and therefore leukocyte cells, esterase orprotease. The corresponding alanine compound (I) is used in the presentday LEUKOSTIX® test strips. ##STR18## The lactate esters of structuralformulae (XI)-(XIV) also were compared to a corresponding alanine ester.

Therefore, the following examples demonstrate the ability of chromogeniclactate esters of structural formula (VI), and particularly ofstructural formula (V), to act as a substrate for HLE, and thereby assaya test sample for leukocyte cells, esterase or protease. It is knownthat amino acid esters, like compound (I), have been used ascolorimetric substrates to detect HLE. The following examples illustratethat the novel, non-amino acid, lactic acid-based colorimetricsubstrates of the present invention are readily hydrolyzed by a serineprotease-based enzyme, like HLE, to provide a detectable anddifferentiable color transition.

EXAMPLE 6

Dry phase test strips incorporating the lactate ester of structuralformula (X), and prepared as discussed above, were compared to dry phasetest strips incorporating the corresponding alanine derivative (I) foran ability to detect HLE in urine. A comparison also was made betweenthe diazonium salt coupling agents DNSA (XX) and NDNSA (XXI). The teststrips were otherwise identical. The results tabulated below wereobtained after quickly immersing the individual reagent strips intostandardized HLE solutions. Two minutes after immersion, the test stripswere examined for a chromogenic response as described above.

    __________________________________________________________________________                         Response at 120 seconds                                               Diazonium                                                                             HLE: 0 ng/mL                                                                           HLE: 19 ng/mL                                   Substrate    Salt    K/S Change                                                                             K/S Change                                      __________________________________________________________________________    Tosyl--Ala--OPP                                                                            DNSA OM 0.021                                                                             --   0.146                                                                             0.125                                       (I)                                                                           Tosyl--Ala--OPP                                                                            NDNSA (XXI)                                                                           0.044                                                                             --   0.308                                                                             0.264                                       (I)                                                                           Tosyl-(L)--Lac--OPP                                                                        NDNSA (XXI)                                                                           0.046                                                                             --   0.348                                                                             0.302                                       (X)                                                                           __________________________________________________________________________

Surprisingly, the lactate ester of structural formula (X) is readilyhydrolyzed by the enzyme HLE, thereby providing a color transition thatis at least as intense as the alanine derivative of structural formula(I). The K/S value and the change in K/S value between 0 and 19 ng/mLHLE are greater for the lactate ester of structural formula (X) than thealanine derivative (I). The lactate ester of structural formula (X)therefore provides a more intense and differentiable color transitionthan the alanine derivative (I) in responding to HLE. In addition, thediazonium salt NDNSA (XXI) provides a larger K/S and a larger change inK/S than the diazonium salt DNSA (XX). Diazonium salts (XXII)-(XXVI)produced a K/S and change in K/S intermediate between diazonium salts(XX) and (XXI). Therefore, the combination of a lactate ester with thediazonium salt NDNSA (XXI) provides an enhanced ability to detect traceto low concentrations of leukocyte cells in a test sample compared tothe presently-used alanine derivative (I).

EXAMPLE 7

Test strips incorporating the lactate ester of structural formula (XI)were compared to test strips incorporating the corresponding alaninederivative in an identical manner as described in Example 6. All teststrips incorporated the diazonium salt NDNSA (XX). The assay results aresummarized below.

    __________________________________________________________________________                      Response at 120 seconds                                                 HLE   R = Alanine                                                                              R = Lactate (X)                                  Compound    (ng/mL)                                                                             K/S  Change                                                                              K/S  Change                                      __________________________________________________________________________    n-Pr--SO.sub.2 --R--OPP                                                                    0    0.050                                                                              --    0.100                                                                              --                                                      19    0.363                                                                              0.313 0.498                                                                              0.398                                                   48    0.881                                                                              0.831 1.049                                                                              0.949                                       __________________________________________________________________________

Similar to Example 6, the lactate ester of structural formula (X)provided a larger change in K/S than the corresponding alaninederivative, therefore providing a more sensitive assay for leukocytecells, esterase or protease by providing a more intense and moredifferentiable color transition.

EXAMPLE 8

Test strips incorporating the lactate ester of structural formula (XII)were compared to test strips incorporating the corresponding alaninederivative in an identical manner as described in Example 6. All teststrips incorporated the diazonium salt NDNSA (XXI). The assay resultsare summarized below.

    __________________________________________________________________________                     Response at 120 seconds                                                 HLE   R = Alanine                                                                              R = Lactate (XI)                                  Compound   (ng/mL)                                                                             K/S  Change                                                                              K/S  Change                                       __________________________________________________________________________    Ph--CO--R--OPP                                                                            0    0.038                                                                              --    0.041                                                                              --                                                      19    0.039                                                                              0.001 0.106                                                                              0.065                                        __________________________________________________________________________

Similar to Examples 6 and 7, the lactate ester of structural formula(XII) provided a larger K/S and a larger change in K/S than thecorresponding alanine derivative. The response for compounds includingthe benzoyl blocking agent, which has a carbonyl group, was not as greatas compounds including a blocking agent having a sulfonyl group. Thislower response is observed in the smaller change in K/S compared to thecompounds tested in Examples 6, 7, 9 and 10. Therefore, alcohol blockinggroups including a sulfonyl group and having the structure (VII) arepreferred over alcohol blocking groups including a carbonyl group andhaving the structure (VIII).

EXAMPLE 9

Test strips incorporating the lactate ester of structural formula (XIII)were compared to test strips incorporating the corresponding alaninederivative in an identical manner as described in Example 6. All teststrips incorporated the diazonium salt NDNSA (XXI). The assay resultsare summarized below.

    __________________________________________________________________________                          Response at 120 seconds                                                  HLE  R = Alanine                                                                            R = Lactate (XII)                              Compound         (ng/mL)                                                                            K/S Change                                                                             K/S  Change                                    __________________________________________________________________________    CH.sub.3 O--CO(CH.sub.2).sub.2 --SO.sub.2 --R--OPP                                              0   0.074                                                                             --   0.177                                                                              --                                                         19   0.435                                                                             0.361                                                                              0.514                                                                              0.337                                                      48   0.976                                                                             0.902                                                                              0.916                                                                              0.739                                     __________________________________________________________________________

The K/S and change in K/S exhibited by the lactate ester of structuralformula (XIII) were similar to the K/S and change in K/S of thecorresponding alanine derivative at low esterase levels, but were lessthan the K/S and change in K/S exhibited by the corresponding alaninederivative at higher esterase levels. However, the K/S and change in K/Sfor the lactate ester of formula (XIII) are sufficient to assay for HLE.

EXAMPLE 10

Test strips incorporating the lactate ester of structural formula (XIV)were compared to test strips incorporating the corresponding alaninederivative in an identical manner as described in Example 6. The assayresults are summarized below.

    __________________________________________________________________________                       Response at 120 seconds                                                  HLE  R = Alanine                                                                             R = Lactate (XI)                                 Compound      (ng/mL)                                                                            K/S  Change                                                                             K/S  Change                                      __________________________________________________________________________    2-Thiophene-SO.sub.2 --R--OPP                                                                0   0.050                                                                              --   0.03 --                                                        19   0.224                                                                              0.174                                                                              0.385                                                                              0.332                                       __________________________________________________________________________

Similar to Examples 6 through 8, the chromogenic lactate ester (XIV)exhibited a larger K/S and a larger change in K/S than the correspondingalanine derivative, therefore providing a more sensitive and accurateassay for leukocyte cells, esterase or protease in a test sample.

As demonstrated in Examples 6-10, chromogenic lactate esters of thepresent invention are capable of differentiating between the absence andpresence of HLE in a test sample and are capable of measuring theconcentration of HLE in a test sample. In addition, the chromogeniclactate esters perform comparably, and typically outperform, HLEsubstrates which contain the conventional amino acids, like the alaninederivative of structural formula (I). Therefore, the lactate esters ofthe present invention represent a new class of compounds useful indetecting leukocyte cells, or elastase or esterase activity, inbiological fluids, such as urine.

Tests also were performed to determine the sensitivity of thechromogenic lactate esters to varying concentrations of HLE. The resultsare tabulated in Table 2, which compares assay results for thechromogenic lactate ester of structural formula (X) to its correspondingalanine derivative. All assays utilized the diazonium salt NDNSA (XXI).

                  TABLE 2                                                         ______________________________________                                        HLE     n-Pr--SO.sub.2 --Ala--OPP                                                                     n-Pr--SO.sub.2 --Lac--OPP                             (ng/mL) ΔK/S.sup.1) (557 mm) at 120 seconds                             ______________________________________                                         5      0.065           0.108                                                  10     0.165           0.257                                                  20     0.353           0.533                                                 100     1.474           1.462                                                 200     1.797           1.693                                                 500     1.628           1.915                                                 ______________________________________                                         .sup.1) change in K/S between 0 ng/mL HLE and the indicated BLE               concentration.                                                           

The alanine derivative can be used to assay for HLE over the range of 5to 500 ng/mL (i.e., about 2 to about 175 cells/μL). Below 5 ng/mL, thecolor transition of the alanine derivative is not sufficiently intenseto provide a differentiable response. In addition, there is nodifference in response to solutions having 200 and 500 ng/mL HLE.Accordingly, a technician cannot readily differentiate between testsample containing 200, 500 or greater than 500 ng/mL of HLE.

However, as illustrated in Table 2, the present lactate esters exhibit arelatively high K/S in the presence of 5 ng/mL HLE, and therefore arecapable of detecting lower levels of HLE in a test sample. In addition,the K/S value for the lactate ester (XI) continues to increase between200 and 500 ng/mL HLE, thereby improving color transitiondifferentiation, e.g., improving instrumental discrimination. Incontrast, when using an alanine derivative, the color formation between200 and 500 ng/mL HLE is sufficiently dark such that a technician wouldhave difficulty differentiating between a test sample containing 200 or500 ng/mL HLE. These results further demonstrate the increasedsensitivity the present chromogenic lactate esters to low concentrationsof HLE, thereby providing an accurate assay for leukocyte cells,esterase or protease over a broader concentration range.

The hydroxy-protected 5-phenyl-3-hydroxy-pyrrolyl-(L)-lactate esters ofthe present invention are a novel class of chromogenic esterasesubstrates having an increased, or comparable, reactivity to thecorresponding 5-phenyl-3-hydroxy-pyrrolyl-tosyl-(L)-alaninatesubstrates, which are presently used in commercial test strip assays forleukocyte cells. The present lactate-based substrates for a serineprotease-based enzyme are also new and advance the art. The presentcomposition, method and device increase leukocyte cell assay sensitivitysuch that as little as 5 ng/mL of HLE can be detected within twominutes, thereby permitting improved detection of trace leukocyte celllevels. The present invention also has the advantage that readtime canbe reduced from 120 to 60 seconds.

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof and therefore only such limitations should be imposed asare indicated by the appended claims.

We claim:
 1. A lactate ester having the structure ##STR19## wherein A isan alcohol blocking group, and wherein B-O- is a residue of a compoundB-OH and provides a detectable response when the lactate ester ishydrolyzed to generate the compound B-OH, wherein the detectableresponse is chromogenic, fluorescent, a reflectance change, a pH change,chemiluminescent, colorimetric, or spectrophotometric.
 2. The ester ofclaim 1 wherein the compound B-OH is selected from the group consistingof a pyrrole, a thiophene, a furan, an azoresorcinol, a phenol, aleukoindophenol, an azo dye,5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3-aceticacid, a 2-substituted-6-hydroxy-benzothiazole having the structure##STR20## wherein: a) at least one of the carbons of positions 4, 5, 6or 7 is linked to a chemical moiety containing an anion group which isattached to the benzene ring; and;b) the carbon at position 2 is linkedto a chemical moiety comprised of at least two atoms which extendresonance of the benzothiazole ring system; and, c) a nitrogen atom islocated at position 3; and d) a sulfur atom is located at position 1, aω-nitrostyryl ester, a resorufin, an acridinone, a merocyanine, an8-hydroxy-2H-dibenz-(b,f)azepin-2-one, a dibenzo azepinone, adibenzothiazepinone, a coumarin ester, and mixtures thereof.
 3. Theester of claim 1 wherein the compound B-OH comprises a pyrrole, athiophene, a furan, or mixtures thereof.
 4. The ester of claim 1 whereinA has a structure ##STR21## wherein R³ is an alkyl group having 3 toabout 22 carbon atoms or R³ is an aryl group.
 5. The ester of claim 1wherein A has a structure ##STR22## wherein R³ is an alkyl group having3 to about 22 carbon atoms or R³ is an aryl group.
 6. The ester of anyone of claims 4 and 5 wherein R³ is selected from the group consistingof phenyl, pyrrolyl, pyridyl, naphthyl, alkyl-substituted phenyl andalkoxy- substituted phenyl, wherein the alkyl or the alkoxy substituenthas one to about 10 carbon atoms.
 7. The ester of claim 1 wherein A isselected from the group consisting of p-toluenesulfonyl,n-propylsulfonyl, benzoyl, carbomethoxyethane sulfonyl, and thiophenesulfonyl.
 8. The ester of claim 1 having the structure ##STR23## whereinA is an alcohol blocking group, X is O,S or NR², R is aryl or loweralkyl, R¹ is hydrogen or lower alkyl, and R² is hydrogen, lower alkyl oraryl.
 9. The ester of claim 8 wherein A is selected from the groupconsisting of p-toluenesulfonyl, n-propylsulfonyl, benzoyl,carbomethoxyethane sulfonyl, and thiophene sulfonyl.
 10. The ester ofclaim 8 wherein X is NR².
 11. The ester of claim 10 wherein R² ishydrogen.
 12. The ester of claim 8 wherein R is phenyl.
 13. The ester ofclaim 8 wherein R¹ is hydrogen.
 14. The ester of claim 1 having thestructure ##STR24## wherein A is selected from the group consisting ofp-toluenesulfonyl, n-propylsulfonyl, benzoyl, carbomethoxyethanesulfonyl, and thiophene sulfonyl.
 15. A composition for determining thepresence or concentration of leukocyte cells, esterase or protease in atest sample, said composition comprising:(a) a lactate ester having thestructure ##STR25## wherein A is an alcohol blocking group, and whereinB-O- is a residue of a compound B-OH, and provides a detectable responsewhen the lactate ester is hydrolyzed to generate the compound B-OH,wherein the detectable response is chromogenic, fluorescent, areflectance change, a pH change, chemiluminescent, colorimetric, orspectrophotometric; (b) a buffer, (c) optionally, an acceleratorcompound capable of increasing the rate of hydrolysis of the lactateester; and (d) optionally, a diazonium salt.
 16. The composition ofclaim 15 comprising an accelerator compound capable of increasing therate of hydrolysis of the lactate ester and a diazonium salt.
 17. Thecomposition of claim 16 wherein the diazonium salt is present in aconcentration of about 0.1 to about 1 millimolar and the acceleratorcompound is present in an amount of about 0.1% to about 2% (v/v) of thecomposition.
 18. The composition of claim 16 wherein the lactate esterhas the structure ##STR26## wherein A is selected from the groupconsisting of p-toluenesulfonyl, n-propylsulfonyl, benzoyl,carbomethoxyethane sulfonyl, and thiophene sulfonyl.
 19. The compositionof claim 15 wherein the compound B-OH is selected from the groupconsisting of a pyrrole, a thiophene, a furan, an azoresorcinol, aphenol, a leukoindophenol, an azo dye,5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3-aceticacid, a 2-substituted-6-hydroxy-benzothiazole having the structure##STR27## wherein: a) at least one of the carbons of positions 4, 5, 6or 7 is linked to a chemical moiety containing an anion group which isattached to the benzene ring; and;b) the carbon at position 2 is linkedto a chemical moiety comprised of at least two atoms which extendresonance of the benzothiazole ring system; and, c) a nitrogen atom islocated at position 3; and d) a sulfur atom is located at position 1, aω-nitrostyryl ester, a resorufin, an acridinone, a merocyanine, an8-hydroxy-2H-dibenz-(b,f)azepin-2-one, a dibenzo azepinone, adibenzothiazepinone, a coumarin ester, and mixtures thereof.
 20. Thecomposition of claim 15 wherein A has a structure ##STR28## wherein R³is an alkyl group having 3 to about 22 carbon atoms or R³ is an arylgroup.
 21. The compound of claim 15 wherein A has a structure ##STR29##wherein R³ is an alkyl group having 3 to about 22 carbon atoms or R³ isan aryl group.
 22. The composition of claim 15 wherein A is selectedfrom the group consisting of p-toluenesulfonyl, n-propylsulfonyl,benzoyl, carbomethoxyethane sulfonyl, and thiophene sulfonyl.
 23. Thecomposition of claim 15 wherein the lactate ester has the structure##STR30## wherein X is O,S or NR², R is aryl or lower alkyl, R¹ ishydrogen or lower alkyl, and R² is hydrogen, lower alkyl or aryl. 24.The composition of claim 23 wherein X is NR².
 25. The composition ofclaim 24 wherein R¹ and R² are hydrogen and R is phenyl.
 26. Thecomposition of claim 25 where A is p-toluenesulfonyl orn-propylsulfonyl.
 27. The composition of claim 15 wherein the lactateester is present in a concentration of about 0.5 to about 2 millimolar.28. The composition of claim 15 wherein the buffer provides a pH ofabout 7 or greater, said pH being in a range wherein the lactate esteris capable of being hydrolyzed.
 29. The composition of claim 15 whereinthe buffer provides a pH of about 7 to about
 10. 30. The composition ofclaim 15 wherein the buffer is present in a concentration of about 200to about 600 millimolar.
 31. The composition of claim 15 wherein theaccelerator compound is present in an amount of 0 to about 4% (v/v) ofthe composition.
 32. The composition of claim 15 wherein the acceleratorcompound comprises an aliphatic alcohol having 3 to about 15 carbonatoms.
 33. The composition of claim 15 wherein the diazonium salt ispresent in a concentration of 0 to about 2 millimolar.
 34. Thecomposition of claim 15 wherein the diazonium salt has the structure##STR31## wherein R⁴, either the same or different, is hydrogen, loweralkyl or aryl, or wherein two adjacent R⁴ groups together form a fusedring system, with the proviso that one of R⁴ is -N⁺ .tbd.N; Y is N orCR⁵, wherein R⁵ is hydrogen or lower alkyl; and D⁻ is an anion.
 35. Thecomposition of claim 15 wherein the diazonium salt has the structure##STR32## wherein F is hydrogen, lower alkyl or hydroxy; D⁻ is acovalently bound anion; G, either the same or different, is hydrogen,lower alkyl or aryl, or both G groups taken together from a fused ringsystem.
 36. The composition of claim 35 wherein the accelerator compoundcomprises an aliphatic alcohol having about 8 to about 15 carbon atoms,and the diazonium salt is selected from the group consisting of1-diazo-2-naphthol-4-sulfonate, 4-diazo-3-hydroxy-7-nitro-1-naphthylsulfonate, and mixtures thereof.
 37. The composition of claim 15 whereinthe diazonium salt is selected from the group consisting of1-diazo-2-naphthol-4-sulfonate, 1-diazophenyl-3-carbonate,4-diazo-3-hydroxy-1-naphthylsulfonate,4-diazo-3-hydroxy-7-nitro-1-naphthylsulfonate,4-diazo-3-hydroxy-1,7-naphthyldisulfonate,2-methoxy-4-(N-morpholinyl)benzene diazonium chloride,4-diazo-3-hydroxy-7-bromo-1-naphthylsulfonate,4-diazo-3-hydroxy-7-cyano-1-naphtylsulfonate,4-diazo-3-hydroxy-7-1-naphthylsulfonate, and mixtures thereof.
 38. Thecomposition of claim 15 further comprising 0% to about 4% by weight of apolymeric material selected from the group consisting ofpolyvinylpyrrolidone, polyvinyl alcohol, gum arabic, gelatin, algin,carrageenan, albumin, a cellulose and mixtures thereof, 0% to about 0.4%by weight of a surfactant, or mixtures thereof.
 39. A composition whichexhibits a sufficient color transition upon contacting a test sample todemonstrate a presence or concentration of leukocyte cells in the testsample, said composition comprising:(a) about 0.5 to about 1.5 mM of achromogenic lactate ester having the structure ##STR33## wherein A isselected from the group consisting of p-toluenesulfonyl,n-propylsulfonyl, benzoyl, carbomethoxyethane sulfonyl, and thiophenesulfonyl; (b) a buffer capable of providing a pH of about 7 to about 10;(c) about 0.1% to about 2% (v/v) of an aliphatic alcohol having about 8to about 15 carbon atoms; and (d) about 0.1 to about 1 mM of a diazoniumsalt coupling agent selected from the group consisting of1-diazo-2-naphthol-4-sulfonate, 1-diazophenyl-3-carbonate,4-diazo-3-hydroxy-1-naphthylsulfonate,4-diazo-3-hydroxy-7-nitro-1-naphthylsulfonate,4-diazo-3-hydroxy-1,7-naphthyldisulfonate,2-methoxy-4-(N-morpholinyl)benzene diazonium chloride,4-diazo-3-hydroxy-7-bromo-1-naphthylsulfonate,4-diazo-3-hydroxy-7-cyano-1-naphthylsulfonate, and4-diazo-3-hydroxy-7-1-naphthylsulfonate, and mixtures thereof.
 40. Amethod of assaying a liquid test sample for the presence orconcentration of leukocyte cells, esterase or protease comprising:(a)contacting the liquid test sample with a reagent composition, saidreagent composition comprising:(i) a lactate ester having the structure##STR34## wherein A is an alcohol blocking group, and wherein B-O- is aresidue of a compound B-OH and provides a detectable response when thelactate ester is hydrolyzed to generate the compound B-OH, wherein thedetectable response is chromogenic, fluorescent, a reflectance change, apH change, chemiluminescent, colorimetric, or spectrophotometric; (ii) abuffer, (iii) optionally, an accelerator compound, which increases therate of hydrolysis of lactate ester, and (iv) optionally, diazonium saltcoupling agent; (b) examining the reagent composition for a detectableresponse; and (c) correlating the detectable response to the presence orconcentration of leukocyte cells, esterase or protease in the liquidtest sample.
 41. The method of claim 40 wherein the test sample isassayed for intact and lysed leukocyte cells.
 42. The method of claim 40wherein the presence or concentration of esterase or protease iscorrelated to the presence or concentration of lysed and intactleukocyte cells.
 43. The method of claim 40 wherein the esterase ishuman leukocyte elastase.
 44. The method of claim 40 wherein the liquidtest sample is urine.
 45. The method of claim 40 wherein the detectableresponse is a chromogenic response.
 46. The method of claim 49 whereinthe reagent composition is examined visually or instrumentally for adetectable response.
 47. The method of claim 40 wherein the reagentcomposition detects and measures an esterase or protease concentrationof about 5 to about 500 nanograms per milliliter of the test sample. 48.The method of claim 47 wherein the esterase or protease concentration iscorrelated to a leukocyte cell concentration of about 2 to about 175cells per microliter of the test sample.
 49. The method of claim 40wherein the reagent composition is examined for a response about oneminute after contacting the test sample.
 50. The method of claim 40wherein the reagent composition comprises an accelerator compound whichincreases the rate of hydrolysis of the lactate ester and a diazoniumsalt coupling agent.
 51. The method of claim 50 wherein the lactateester has the structure ##STR35## wherein X is O,S or NR², R is aryl orlower alkyl, R¹ is hydrogen or lower alkyl, and R² is hydrogen, loweralkyl or aryl.
 52. The method of claim 51 wherein the lactate ester hasthe structure ##STR36## wherein A is selected from the group consistingof p-toluenesulfonyl, n-propylsulfonyl, benzoyl, carbomethoxyethanesulfonyl, and thiophene sulfonyl.
 53. The method of claim 52 wherein theaccelerator compound comprises an aliphatic alcohol having about 8 toabout 15 carbon atoms, and the diazonium salt coupling agent is selectedfrom the group consisting of 1-diazo-2-naphthol-4-sulfonate,4-diazo-3-hydroxy-7-nitro-1-naphthyl sulfonate, and mixtures thereof.54. The method of claim 40 wherein the assay for the presence orconcentration of leukocyte cells, esterase or protease is a dry phaseassay.